Transcriptomic and Physiological Analyses Reveal Potential Genes Involved in Photoperiod-Regulated ß-Carotene Accumulation Mechanisms in the Endocarp of Cucumber (Cucumis sativus L.) Fruit.

The accumulation of carotenoids in plants is a key nutritional quality in many horticultural crops. Although the structural genes encoding the biosynthetic enzymes are well-characterized, little is known regarding photoperiod-mediated carotenoid accumulation in the fruits of some horticultural crops. Herein, we performed physiological and transcriptomic analyses using two cucumber genotypes, SWCC8 (XIS-orange-fleshed and photoperiod-sensitive) and CC3 (white-fleshed and photoperiod-non-sensitive), established under two photoperiod conditions (8L/16D vs. 12L/12D) at four fruit developmental stages. Day-neutral treatments significantly increased fruit ß-carotene content by 42.1% compared to short day (SD) treatments in SWCC8 at 40 DAP with no significant changes in CC3. Day-neutral condition elevated sugar levels of fruits compared to short-day treatments. According to GO and KEGG analyses, the predominantly expressed genes were related to photosynthesis, carotenoid biosynthesis, plant hormone signaling, circadian rhythms, and carbohydrates. Consistent with ß-carotene accumulation in SWCC8, the day-neutral condition elevated the expression of key carotenoid biosynthesis genes such as PSY1, PDS, ZDS1, LYCB, and CHYB1 during later stages between 30 to 40 days of fruit development. Compared to SWCC8, CC3 showed an expression of DEGs related to carotenoid cleavage and oxidative stresses, signifying reduced ß-carotene levels in CC3 cucumber. Further, a WGCNA analysis revealed co-expression between carbohydrate-related genes (pentose-phosphatase synthase, ß-glucosidase, and trehalose-6-phosphatase), photoperiod-signaling genes (LHY, APRR7/5, FKF1, PIF3, COP1, GIGANTEA, and CK2) and carotenoid-biosynthetic genes, thus suggesting that a cross-talk mechanism between carbohydrates and light-related genes induces ß-carotene accumulation. The results highlighted herein provide a framework for future gene functional analyses and molecular breeding towards enhanced carotenoid accumulation in edible plant organs.

Genome-Wide Identification of the B-Box Gene Family and Expression Analysis Suggests Their Potential Role in Photoperiod-Mediated ß-Carotene Accumulation in the Endocarp of Cucumber (Cucumis sativus L.) Fruit.

Carotenoids are indispensable to plants and essential for human nutrition and health. Carotenoid contents are strongly influenced by light through light-responsive genes such as B-Box (BBX) genes. BBX proteins, a class of zinc-finger transcription factors, mediate many light-signaling pathways, leading to the biosynthesis of important metabolites in plants. However, the identification of the BBX gene family and expression analysis in response to photoperiod-mediated carotenoid accumulation in cucumber remains unexplored. We performed a genome-wide study and determined the expression of cucumber BBX genes (hereafter referred to as CsaBBXs genes) in the endocarp of Xishuangbanna cucumber fruit (a special type of cucumber accumulating a high level of ß-carotene in the endocarp) using an RNA-seq analysis of plants previously subjected to two photoperiodic conditions. Here, 26 BBX family genes were identified in the cucumber genome and named serially CsaBBX1 through CsaBBX26. We characterized CsaBBX genes in terms of their phylogenetic relationships, exon-intron structures, cis-acting elements, and syntenic relationships with Arabidopsis thaliana (L.) Heynh. RNA-seq analysis revealed a varied expression of CsaBBX genes under photoperiod treatment. The analysis of CsaBBXs genes revealed a strong positive correlation between CsaBBX17 and carotenoid biosynthetic pathway genes (phytoene synthase, ζ-carotene desaturase, lycopene ε-cyclase, ß-carotene hydroxylase-1), thus suggesting its involvement in ß-carotene biosynthesis. Additionally, nine CsaBBX genes (CsaBBX 4,5,7,9,11, 13,15,17 and 22) showed a significant positive correlation with ß-carotene content. The selected CsaBBX genes were verified by qRT-PCR and confirmed the validity of RNA-seq data. The results of this study established the genome-wide analysis of the cucumber BBX family and provide a framework for understanding their biological role in carotenoid accumulation and photoperiodic responses. Further investigations of CsaBBX genes are vital since they are promising candidate genes for the functional analysis of carotenoid biosynthesis and can provide genetic tools for the molecular breeding of carotenoids in plants.

A novel mutation in TFL1 homolog sustaining determinate growth in cucumber (Cucumis sativus L.).

KEY MESSAGE: BSA-seq combined with whole-genome resequencing map-based cloning delimited the cucumber det-novel locus into a 44.5 kb region in chromosome 6 harboring a putative candidate gene encoding a phosphatidylethanolamine-binding protein (CsCEN). Determinate and indeterminate growth habits of cucumber can affect plant architecture and crop yield. The TERMINAL FLOWER 1 (TFL1) controls determinate/indeterminate growth in Arabidopsis. In this study, a novel mutation in cucumber TFL1 homolog (CsCEN) has shown to regulate determinate growth and product of terminal flowers in cucumber (Cucumis sativus L.), which is similar to the function of CsTFL1 as previously reported. Genetic analysis in two determinate genotypes (D226 and D082) and indeterminate genotype (CCMC) revealed that a single recessive gene is responsible for this determinate growth trait. With the combination of BSA-seq and whole-genome resequencing, the locus of determinate-novel (det-novel) trait was mapped to a 44.5 kb genomic region in chromosome 6. Sequence alignment identified one non-synonymous SNP mutation (A to T) in the third exon of CsCEN, resulting in an amino acid substitution (Thr to Pro), suggesting that determinate growth might be controlled by a novel gene CsCEN (Csa6G152360) which differed from the reported CsTFL1 gene. The CsCEN expression level in shoot apexes and axillary buds was significantly lower in D226 compared to CCMC, suggesting its essential role in sustaining indeterminate growth habit. Identification and characterization of the CsCEN in the present study provide a new insight into plant architecture modification and development of cucumber cultivars suited to mechanized production system.

Brassinosteroids induce plant tolerance against phenanthrene by enhancing degradation and detoxification in Solanum lycopersicum L.

Polycyclic aromatic hydrocarbons (PAHs) are toxic to both plants and animals. The enhancement of plant tolerance and detoxification capacity is important for the plant-based remediation of PAHs. Therefore, we investigated the effects of 24-epibrassinolide (EBR) on the metabolism of a three-ringed PAH (phenanthrene-PHE) and subsequent stress tolerance in tomato (Solanum lycopersicum L.) plants. Exposure to PHE (300 µM) for 21 d significantly decreased biomass and net CO(2) assimilation (P(n)) but induced photoinhibition, malondialdehyde (MDA), H(2)O(2) and antioxidant enzymes. Obvious ultrastructural alterations were observed in the PHE-treated root tip cells. Importantly, the foliar application of EBR (0.1 µM) significantly increased biomass, P(n) and antioxidant enzyme activities but decreased MDA and H(2)O(2) compared with PHE alone and saved the root cells from severe damage. The expression of detoxification genes (CYP90b3, GSH1, GST1), reduced glutathione (GSH) content and glutathione S-transferase activity in the EBR+PHE-treated plants were higher than those of PHE alone. Additionally, lower levels of PHE residues in the roots were observed as a result of EBR+PHE treatment. Taken together, our results strongly suggest an enhanced and coordinated detoxification and degradation of PHE by EBR.

Brassinosteroid alleviates phenanthrene and pyrene phytotoxicity by increasing detoxification activity and photosynthesis in tomato.

The present study was carried out to investigate the effects of exogenously applied 24-epibrassinolide (BR) on growth, gas exchange, chlorophyll fluorescence characteristics, lipid peroxidation and antioxidant systems of tomato seedlings grown under different levels (0, 10, 30, 100 and 300µM) of phenanthrene (PHE) and pyrene (PYR) in hydroponics. A concentration-dependent decrease in growth, photosynthetic pigment contents, net photosynthetic rate (Pn), stomatal conductance (Gs), maximal quantum yield of PSII (Fv/Fm), effective quantum yield of PSII (Φ(PSII)), photochemical quenching coefficient (qP) has been observed following PHE and PYR exposure. By contrast, non-photochemical quenching coefficient (NPQ) was increased. PHE was found to induce higher stress than PYR. However, foliar or root application of BR (50nM and 5nM, respectively) alleviated all those depressions with a sharp improvement in the activity of photosynthetic machinery. The activities of guaicol peroxidase (GPOD), catalase (CAT), ascorbate peroxidase (APX) and glutathione reductase (GR) as well as content of malondialdehyde (MDA) were increased in a dose-dependent manner under PHE or PYR treatments. Compared with control the highest increments of GPOD, CAT, APX, GR and MDA by PHE/PYR alone treatments were observed following 300µM concentration, which were 67%, 87%, 53%, 95% and 74% by PHE and 42%, 53%, 30%, 86% and 62% by PYR, respectively. In addition, both reduced glutathione (GSH) and oxidized glutathione (GSSG) were induced by PHE or PYR. Interestingly, BR application in either form further increased enzymatic and non enzymatic antioxidants in tomato roots treated with PHE or PYR. Our results suggest that BR has an anti-stress effect on tomato seedlings contaminated with PHE or PYR and this effect is mainly attributed by increased detoxification activity.

Response of ascorbate peroxidase isoenzymes and ascorbate regeneration system to abiotic stresses in Cucumis sativus L.

Ascorbate peroxidase (APX) isoenzymes, distributing in at least four distinct cell compartments, the chloroplastic stroma (sAPX) and thylakoid membrane (tAPX), microbody (mAPX) and cytosol (cAPX), catalyze the reduction of H(2)O(2) to water by using ascorbic acid (AsA) as specific electron donor. In order to better clarify the response of APX isoenzymes and AsA regeneration enzymes to abiotic stresses, the activities of APX isoenzymes as well as monodehydroascorbate reductase (MDAR), glutathione reductase (GR) and dehydroascorbate reductase (DHAR) were investigated in cucumber plants after heat, methyl viologen (MV) and H(2)O(2) treatments. The activities of cAPX, sAPX, mAPX increased after a slight decline throughout the experiment. Consistent closely with sAPX activity, the expression of sAPX followed a similar change pattern, indicating that sAPX was regulated at the transcriptional level. In contrast, constitutive expression was observed in tAPX activity and no significant changes in tAPX activity were found throughout the experiment. The increases in MDAR and GR were accompanied with enhanced level of AsA/DHA, implying that the AsA regeneration system plays an essential role in compensating AsA degradation.